This invention is directed to the fields of molecular biology and medicine.
Recombinant DNA technology has made it possible to produce large amounts of highly purified proteins. Many of these proteins are now in use as pharmaceuticals. Recombinantly produced pharmaceutical proteins include, for example, insulin, erythropoietin, human growth hormone and β-interferon.
Pharmaceutical proteins which need to gain systemic access cannot be administered enterally because the enzymes of the digestive system degrade the proteins before they gain access. Therefore, pharmaceutical proteins generally are administered by injection. Diseases that require repeated administration of a protein over a long period of time, such as diabetes, can require daily injection. Of course, frequent injections are not pleasant for the patient and may not be the best method of administration. Therefore, means to deliver proteins without injection would provide an advantage.
Various proteins are known to gain access to the system by traversing mucosal surfaces. For example, van Deurs et al. (European J. Cell Biol., 51:96 (1990)) showed that ricin crosses the epithelium by transcytosis. EP 0 222 835 B1 (Russell-Jones et al. May 25, 1987) discusses the use of carrier molecules that specifically interact with the mucosal epithelium, including various toxins, for the oral delivery of immunogens for inducing cell-mediated immunity.
Pseudomonas exotoxin A (PE) is a toxic protein produced by the bacterium, Pseudomonas aeruginosa. In its native form, the protein binds to the α2-macroglobulin receptor (“α2-MR”) which is found on the surface of many cells. The molecule comprises four domains. Domain la binds α2-MR. Domain II is responsible for endocytosis of the molecule into the cell. Domain Ib has no identified function. Domain III is responsible for toxicity (by mediating inactivation of protein synthesis) and acts to retain the toxin in the endoplasmic reticulum. PE has been extensively re-engineered to give the molecule new properties. For example, domain la has been replaced with proteins that bind to specific target receptors. Targeting proteins also have been engineered into domain III to provide a binding capability. Such constructs have found use as immunotoxins. Domain III has been modified to eliminate the ADP ribosylation activity. Domain II has been shortened while retaining translocation ability. Thus, the domains of PE act as relatively independent functional units which can be exchanged for other functional units and that can be extensively engineered within themselves. See, for example, U.S. Pat. No. 5,863,745 (FitzGerald et al.); U.S. Pat. No. 5,854,044 (Pastan et al.); U.S. Pat. No. 5,705,163 (Pastan et al.); U.S. Pat. No. 5,705,156 (Pastan et al.); U.S. Pat. No. 5,696,237 (FitzGerald et al.); U.S. Pat. No. 5,602,095 (Pastan et al.); U.S. Pat. No. 5,458,878 (Pastan et al.); U.S. Pat. No. 5,082,927 (Pastan et al.); U.S. Pat. No. 4,892,827 (Pastan et al.); Y. Reiter et al. Nature Biotechnology (1996) 14:1239 and U. Brinkmann and I. Pastan, Biochim. et Biophys. Acta (1994) 1198:27.